Various methods such as biosynthesis, chemical synthesis and cell-free synthesis are known to be used for synthesizing proteins. In the biosynthesis method, a protein is obtained by utilizing the inside of a cell such as an Escherichia coli cell and introducing and expressing DNA encoding the protein to be synthesized in the cell. In the chemical synthesis, the objective protein is synthesized by sequentially binding amino acids in an organochemical manner. In the cell-free synthesis, the protein is synthesized in a cell-free system utilizing an enzyme, etc., present in various cells such as the Escherichia coli cell. These methods are appropriately used separately or combined depending on the intended use, the size and the nature to be added, of the protein.
At present, in order to synthesize a protein homogeneously having a particular modification with a sugar chain or a lipid, etc., in a middle part of its amino acid sequence, amino acids are modified with the sugar chain or the lipid, etc., in advance and then a peptide chain including the modified amino acids is chemically synthesized.
A solid-phase synthesis is mainly used as the method for chemically synthesizing the peptide chain. However, the peptide chain obtained by the solid-phase synthesis is generally a short chain, and is composed of at longest about 50 residues.
Thus, the short peptide chains are separately prepared and then they are ligated in order to synthesize the long peptide chain having the modification. Various techniques for ligating the peptide chains have been reported, and one widely used technique is the native chemical ligation method (NCL method). The NCL method can also be applied between unprotected peptide chains, and is known to be useful for forming a native amide bond (peptide bond) at a ligation site (e.g., Patent Literature 1). The NCL method is a chemoselective reaction between a first peptide having an α-carboxythioester moiety at its C-terminal and a second peptide having a cysteine residue at its N-terminal, and a thiol group (SH group, also referred to as a sulfhydryl group) of the cysteine side chain is selectively reacted with carbonyl carbon of a thioester group, whereby a thioester binding initial intermediate is formed by the thiol exchange reaction. This intermediate spontaneously performs intramolecular transposition to give the native amide bond at the ligation site while it regenerates the cysteine side chain thiol.
In this method, two peptide chains can be ligated via the peptide bond only by mixing the unprotected peptides in a buffer solution. In the NCL method, even when compounds such as peptides having many functional groups are reacted, the C-terminal of one peptide can be ligated selectively to the N-terminal of the other peptide. From these points, it is important to determine in what way to utilize the NCL method in order to chemically synthesize the protein.
However, a problem when the NCL method is utilized includes the preparation of a peptide thioester having an α-carboxythioester moiety at its C-terminal, which is required as a raw material. Various methods have been reported for preparing the peptide thioester, and those methods can be generally classified into two types based on the solid-phase synthesis.
A first one is the method of constructing the peptide thioester on a resin. In this method, the peptide thioester can be obtained together with cleavage of the peptide chain from the resin after constructing the peptide (e.g., Boc solid-phase synthesis, Fmoc solid-phase synthesis). A second one is the method of constructing the peptide chain on the solid phase via a linker equivalent to thioester (Safety catch linker, Fujii method, Dawson method, Mercapto propanol method, Kawakami method, Danishefsky method, Hojo method, Aimoto method, etc.). In this method, thioester is obtained by activating the peptide chain C-terminal constructed by appropriately treating with the linker, followed by thiolysis of the peptide chain (Non-patent Literature 1).
In addition to these methods, the method in which a protected peptide so that the side chain is protected by the solid-phase synthesis and only the carboxyl group at the C-terminal is free is synthesized followed by thioesterification under an appropriate condensation condition has also been reported (e.g., Patent Literature 2). Any of these methods have been well-established, and used for various protein syntheses. However, the size of the peptide thioester capable of being synthesized is limited because these methods are limited to restrictions of the solid-phase synthesis. Further, in the method using the linker, a non-native amino acid derivative or a specific derivative must be chemically synthesized separately. Thus, their procedures cannot always be said to be simple.
An intein method solved the restriction of the thioesterification by the solid-phase synthesis (Non-patent Literature 2). In this method, a polypeptide fragment biosynthesized from a cell can be obtained as thioester. In the intein method, the peptide chain is thioesterified by utilizing a protein splicing function that occurs in the particular protein sequence, and the polypeptide chain is obtained as thioester. An advantage of this method is that a long chain peptide thioester can be obtained. The synthesis of the large modified protein, which had been considered to be difficult to synthesize until now, has become possible by combining this method with the chemical synthesis method (Non-patent Literature 3). The method of expressing the polypeptide chain and obtaining it has been studied extensively, and well-established as a basic technique in biology.
However, when the intein method is used, a peptide sequence to be targeted is necessary and an expressed intein complex protein must be folded to take on an inherent three-dimensional structure because not only is the polypeptide expressed but also the protein splicing is caused to function. Thus, depending on the polypeptide sequence to be expressed, the peptide thioester is not always obtained in association with sufficient conditions for optimization and the accompanying complications in the work.
Meanwhile, the method of cleaving the peptide chain at a position of a cysteine residue by reacting a compound with the SH group of the cysteine residue in the peptide (Non-patent Literatures 4 and 5), and the method of cleaving the peptide bound to the solid phase using the linker (Non-patent Literatures 6 and 7) are known as the methods of cleaving the peptide. Also, the method of cleaving the peptide bond on the C-terminal-side of a methionine residue using cyanogen bromide (CNBr) is known. However, these are not methods for obtaining a peptide fragment as the thioester.